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Drosophila importin-7 functions upstream of the Elmosignaling module to mediate the formation andstability of muscle attachments

Ze Cindy Liu1,2, Nadia Odell1 and Erika R. Geisbrecht1,*1Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri, Kansas City, MO 64110, USA2PhD Program, School of Biological Sciences, University of Missouri, Kansas City, MO 64110, USA

*Author for correspondence (geisbrechte@umkc.edu)

Accepted 30 August 2013Journal of Cell Science 126, 5210–5223� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.132241

SummaryEstablishment and maintenance of stable muscle attachments is essential for coordinated body movement. Studies in Drosophila havepioneered a molecular understanding of the morphological events in the conserved process of muscle attachment formation, including

myofiber migration, muscle–tendon signaling, and stable junctional adhesion between muscle cells and their corresponding targetinsertion sites. In both Drosophila and vertebrate models, integrin complexes play a key role in the biogenesis and stability of muscleattachments through the interactions of integrins with extracellular matrix (ECM) ligands. We show that Drosophila importin-7 (Dim7)

is an upstream regulator of the conserved Elmo–MbcRRac signaling pathway in the formation of embryonic muscle attachment sites(MASs). Dim7 is encoded by the moleskin (msk) locus and was identified as an Elmo-interacting protein. Both Dim7 and Elmo localizeto the ends of myofibers coincident with the timing of muscle–tendon attachment in late myogenesis. Phenotypic analysis of elmo

mutants reveal muscle attachment defects similar to those previously described for integrin mutants. Furthermore, Elmo and Dim7interact both biochemically and genetically in the developing musculature. The muscle detachment phenotype resulting from mutationsin the msk locus can be rescued by components in the Elmo signaling pathway, including the Elmo–Mbc complex, an activated Elmovariant, or a constitutively active form of Rac. In larval muscles, the localization of Dim7 and activated Elmo to the sites of muscle

attachment is attenuated upon RNAi knockdown of integrin heterodimer complex components. Our results show that integrins functionas upstream signals to mediate Dim7–Elmo enrichment to the MASs.

Key words: Drosophila, Muscle, Cell adhesion, Elmo, Dim7, Myotendinous junction

IntroductionThe Drosophila body wall musculature, which is functionally and

structurally similar to vertebrate skeletal muscles, is a usefulmodel to study conserved, complex developmental processes,including cell fusion, cell migration and cell–extracellular matrix

(ECM) adhesion. The muscle–tendon attachment site, ormyotendinous junction (MTJ), is a highly-specialized cell–ECM adhesion site, through which the ends of striated muscles

are connected to specialized epidermal attachment cells calledtendon cells (Brown, 2000; Gilsohn and Volk, 2010).Functionally, the proper establishment and maintenance ofmuscle attachment sites (MASs) in Drosophila embryogenesis

is essential for tendon cells to withstand mechanical forcesgenerated by contracting muscles (Volk, 1999; Schweitzer et al.,2010; Schejter and Baylies, 2010).

Structurally, integrin-mediated hemi-adherens junctionsprovide a crucial link in stable MAS formation. Thetransmembrane integrin heterodimer complex on the surface of

both muscle (aPS2bPS) and tendon (aPS1bPS) cells anchorsthese opposing cell types to the adjacent ECM by binding to theligands Tiggrin (Tig), Laminin (Lam) or Thrombospondin (Tsp)

(Bunch et al., 1998; Graner et al., 1998; Chanana et al., 2007;Subramanian et al., 2007). Internally, a core group of proteinsassociate with the cytoplasmic tail of integrins to bridge the

link between the extracellular milieu and the internal actin

cytoskeleton, including the IPP complex [integrin-linked kinase(ILK), PINCH and Parvin], Talin and Wech (Clark et al., 2003;

Loer et al., 2008; Zervas et al., 2001; Legate et al., 2006; Sebe-

Pedros et al., 2010; Vakaloglou and Zervas, 2012). Focaladhesion kinase (FAK) and Git1 are two additional proteins

that accumulate at the MASs in late embryonic myogenesis, themolecular function of which is unclear (Bahri et al., 2009;

Grabbe et al., 2004). We recently showed that mutations in the

msk locus result in the detachment of muscles from theircorresponding tendon cells in late embryogenesis (Liu and

Geisbrecht, 2011).

The Drosophila Engulfment and Cell Motility (Elmo)–

Myoblast city (Mbc)RRac signaling pathway is evolutionarily

conserved from Caenorhabditis elegans to vertebrates (ELMO–DOCK180) and is essential for many developmental processes,

including phagocytosis and cell migration (Katoh and Negishi,2003; Cote and Vuori, 2007). A diverse array of receptors across

multiple organisms have been identified upstream of Elmo–Dock

complexes that regulate Rac activation. Integrins, growth factorreceptors and phagocytic receptors are responsible for relaying

extracellular signals to internal ELMO–DOCK signaling

complexes to promote cell migration, phagocytosis or neuriteoutgrowth in mammalian systems (Cote and Vuori, 2007;

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Kinchen and Ravichandran, 2007; Miyamoto and Yamauchi,2010; Park and Ravichandran, 2010). Studies in C. elegans have

revealed the INA-1 integrin a-subunit, transmembrane proteinCED-1, and the phosphatidylserine receptor function asengulfment receptors in apoptotic cell clearance (Hsu and Wu,2010; Kinchen et al., 2005; Wang et al., 2003). However, in

Dictyostelium discoideum, ElmoE has been identified as anessential component to mediate G-protein-coupled receptor(GPCR) signaling to a Dock–RacB complex (Yan et al., 2012).

The functional conservation of the Elmo–MbcRRac signalingpathway in Drosophila is well-established in multiple biologicalprocesses, including ommatidial development, myoblast fusion

and cell migration; although little is known about the upstreamsignals that regulate the ElmoRRac complex (Luo et al., 1994;Duchek et al., 2001; Hakeda-Suzuki et al., 2002; Geisbrecht et al.,2008; Bianco et al., 2007; Nolan et al., 1998). Platelet-derived

growth factor- and vascular endothelial growth factor-receptorrelated (PVR) is known to act upstream of Rac in border cellmigration and thorax closure, but the upstream receptor in eye

and muscle cells remain elusive (Ishimaru et al., 2004; Ducheket al., 2001; Bianco et al., 2007). The existence of a constitutiveinteraction between endogenous Dock180 and Elmo is observed

in CHO cells, regardless of the presence of extracellular stimuli(Patel et al., 2011), suggesting that there are yet, unidentifiedsignals or proteins that regulate the activity of this complex.

Elmo comprises N-terminal Armadillo repeats, an internally

conserved ‘ELMO’ domain with unknown function, and C-terminal pleckstrin homology (PH) and proline-rich (PxxP)motifs that directly bind to Dock family members (Patel et al.,

2010). A combination of bioinformatics approaches and in vitro

validation has revealed structural domains that regulate Elmoactivity through an intracellular autoinhibitory switch (Patel et al.,

2010). An N-terminal Elmo inhibitory domain (EID), composedof HEAT and Armadillo repeats, physically interacts with anElmo autoregulatory domain (EAD) residing between the PH and

PxxP motifs in the C-terminus. Mutations in ELMO1 thatdisrupt the direct EAD–EID binding result in an active,open conformation. When expressed in integrin-activated cells,activated Elmo mutants (ELMO1 I204D or ELMO1 M692A/

E693A) accumulate at the cell periphery to promote cellelongation and cell motility (Patel et al., 2010). The associationof active RhoG with ELMO1 competes with the endogenous

EID–EAD interaction, or closed conformation, within ELMO1.Thus, after binding to ELMO1, RhoG may unleash the open,active form of Elmo or recruit the closed conformation of Elmo

to the membrane. Drosophila does not have a RhoG ortholog andthe GTPase Rac exhibits the highest homology by primarysequence comparison. Thus, the identification of extracellular

triggers and upstream molecules that mediate Elmo–Dock mutualautoinhibition and Rac activation in vivo remains unclear.

In this manuscript, we demonstrate a novel role for Elmo in theformation and maintenance of MASs. Furthermore, we place

Dim7 as an upstream component of the canonical Elmo–MbcRRac signaling pathway and we show that integrinsregulate the subcellular distribution of Dim7 and activated

Elmo to the ends of actively contracting muscles. We postulatethat the intracellular temporal and spatial regulation of Racactivity at the muscle membrane serves to locally remodel the

actin cytoskeleton to modulate the stability of the muscle–tendonjunctions during muscle growth or in response to changes in forcetransmission in active muscle contraction.

ResultsWe previously identified the Dock family members Mbc and

Sponge (Spg) in an in vivo proteomics approach aimed atuncovering proteins that physically interact with Elmo inDrosophila embryogenesis (Geisbrecht et al., 2008; Biersmith

et al., 2011). Herein, we present an additional Elmo-interactingprotein that emerged from this mass spectrometry (MS) screen(Geisbrecht et al., 2008). The Drosophila Importin-7 ortholog

(Dim7), encoded by the moleskin (msk) locus, was present inseven Elmo HA-tagged immunoprecipitates versus one untaggedElmo run. MS analysis after isolation of Mbc–HA complexes

also revealed Dim7 peptides in six individual Mbc-tagged runsversus none in untagged controls.

Mutations in elmo and the rac genes exhibit muscledetachment phenotypes

Based upon the identification of Dim7 as a potential Elmo-

binding candidate and our previous characterization of msk inmuscle attachment (Liu and Geisbrecht, 2011), we began ouranalysis by examining elmo mutant embryos for myogenic

defects after the completion of myoblast fusion. In allexperiments except where noted, we focused on the ventralmusculature, where the muscles have a stereotypical rectangular

shape and the attachment sites are relatively large in wild-type(WT) embryos (Fig. 1A,A9). Next, we analyzed embryos trans-heterozygous for a particular combination of elmo alleles(elmo19F3/KO; Fig. 1B,B9) or elmo germline clone (GLC)

embryos (Fig. 1C,C9), in which the maternal and zygoticcontribution of a hypomorphic elmo allele (elmoPB[c06760]) wasdepleted. Although the myoblast fusion defects varied in severity

depending upon the genotype (Geisbrecht et al., 2008), thenumber of detached muscles was comparable in elmo zygotic orGLC embryos. Furthermore, a decrease in Elmo protein levels

was not biased towards any particular muscle groups, suggestingthat Elmo is required in all muscles for proper muscleattachment.

All available alleles of mbc result in severe myoblast fusiondefects (Erickson et al., 1997; Balagopalan et al., 2006). Thistechnical limitation precluded phenotypic analysis of Mbc

function in later myogenic events, including MAS formation.To further test whether other components of the Elmo–MbcRRac pathway are essential in muscle–tendon attachment,we took two approaches to reduce Rac activity in the developing

muscle. Even though both Rac1 and Rac2 in Drosophila areessential for actin-mediated myoblast fusion (Hakeda-Suzukiet al., 2002), small amounts of muscle fibers are able to form in

rac1rac2 mutant embryos because of the perdurance ofmaternally loaded gene product (Fig. 1D,D9). These myofibersround up upon muscle contraction, similar to the muscle

detachment phenotype observed in elmo or msk mutants.Furthermore, expression of dominant-negative Rac (RacN17) inventral longitudinal muscle 1 (VL1, also known as muscle 12)

using the 5053-GAL4 driver resulted in detached muscles(supplementary material Fig. S1). This data, taken together,demonstrate that both Elmo and the Rac proteins are essential forembryonic muscle attachment.

To determine whether the Elmo–Dim7 complex is requiredfor maintaining attachment in mature contractile muscles, we

utilized RNAi approaches to examine the requirement for Dim7,Elmo and Mbc in the larval musculature. Expression of UAS-RNAi constructs to knockdown elmo, msk or mbc levels with

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mef2-GAL4 resulted in pupal lethality (data not shown). Fillets of

third larval (L3) instar control animals (mef2-GAL4/+) exhibited

a normal muscle pattern. Upon a reduction in Elmo, Dim7 or

Mbc levels, dissected L3 individuals revealed occasional muscle

detachment accompanied by frequent missing muscles and

muscle fiber thinning (supplementary material Fig. S1). Thus,

Dim7 and the Elmo–Mbc complex all play essential roles in the

MAS maintenance from late embryonic myogenesis through to

larval stages.

The inability of the somatic muscles to remain attached in elmo

mutant embryos raised the possibility that abnormal integrin-

mediated adhesion is an underlying cause of muscle detachment.

To further elucidate the role of Elmo in embryonic muscle

attachment, the protein distribution of known MAS and tendon

cell markers were examined. In WT embryos, both bPS integrin

(Fig. 1E,E9) and the ECM ligand Tig (Fig. 1F,F9) accumulated at

the interface between the muscle and tendon cells. In zygotic

elmo mutants, bPS integrin (Fig. 1I,I9) or Tig (Fig. 1J,J9) were

correctly localized to the hemi-adherens junctions at the segment

borders, although the MASs were reduced in size. Owing to the

phenotypic similarities of the muscle attachment defects in both

elmo and msk mutants (Liu and Geisbrecht, 2011), we chose to

further examine whether other proteins known to be affected in

msk mutants were similarly aberrant upon loss of Elmo.

Previous cell-specific rescue experiments revealed that Dim7

exhibits both muscle-cell autonomous and non-autonomous roles,

where Dim7 is responsible for the localization of phosphorylated

FAK (pFAK) in the muscle cell and regulates the nuclear

translocation of phosphorylated MAPK (pMAPK) and Sr for the

maintenance of tendon cell identity through the secreted Vein–Egfr

Fig. 1. Mutations affecting components of the ElmoRRac signaling pathway are defective in somatic muscle attachment. (A–P) The ventral musculature

in stage 16–17 embryos stained with anti-myosin heavy chain (MHC; A–D9, M–P) or anti-tropomyosin (TM; E–L0). (A,A9) The stereotypical muscle pattern in

WT embryos. (B–C9) Detached muscle fibers (arrows) are present in elmo19F3/KO zygotic (B,B9) or elmo GLC embryos (C,C9). (D,D9) Embryos mutant for rac1, rac2

exhibit muscle attachment defects (arrows). (E–K9) The localization of attachment site proteins (green; arrowhead) in two hemisegments of the ventral musculature

(white). (E–F9, I–J9) bPS-integrin and Tig are properly localized to MASs in both WT (E,F9) and elmo mutant embryos (I–J9). (G–K9) Phosphorylated FAK

(pFAK) is present at the ends of muscle fibers in both WT (G,G9) and elmo mutants (K,K9). (H–H-,L–L-) pMAPK normally accumulates in tendon cell nuclei.

Loss of Elmo results in a decrease in pMAPK-positive tendon cells (L,L0) compared with WT embryos (H,H9). (M–P) Representative images of ventral muscle

attachment phenotypes scored in cell-specific rescue experiments: WT (M); WT-like normal muscle pattern with a few unfused myoblasts (N); minor muscle

attachment defects (O); or severe muscle attachment defects (P). (Q) Graph quantifying the extent of muscle or tendon cell-specific rescue in elmo19F3/KO mutants.

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signaling pathway (Liu and Geisbrecht, 2011). To determinewhether Elmo similarly affects pFAK and/or pMAPK proteins,

both WT and elmo mutant embryos were immunostained toexamine the subcellular localization and levels of these proteins. Incontrast to msk mutants where pFAK protein is not detectable (Liuand Geisbrecht, 2011), this phosphoprotein was found to

accumulate at the MASs in both WT (Fig. 1G,G9) and elmo

(Fig. 1K,K9) mutant embryos. pMAPK is normally localized to thetendon cell nuclei in WT embryos (ref; Fig. 1H,H9) but this

accumulation was reduced in elmo mutants (Fig. 1L,L9). Thus, areduction in both elmo and msk levels results in loss of pMAPK,although it is less obvious in elmo mutants, consistent with the

weaker muscle detachment phenotypes observed in zygotic elmo

mutants. In summary, loss of Elmo or Dim7 result in similar muscledetachment phenotypes and a decrease in the number of maturetendon cells as determined by pMAPK staining.

Based upon the final muscle morphology observed inhomozygous elmo mutant embryos, we categorized the detachedmuscles into three phenotypic groups. Class I embryos (WT or

WT-like) had rectangular muscles connected to MASs with a largeinterface area between muscles and their target tendon cells(Fig. 1M). WT-like embryos retained normal muscle attachment

with occasional unfused myoblasts (Fig. 1N). Moderate defectswere observed in elmo mutants designated as Class II, in whichspindle-shaped muscles with pointed ends exhibited small MASs

and weakened muscle attachment (Fig. 1O). Mutants in Class IIIexhibited severe defects, with rounded muscles and a completeloss of muscle attachment to the epidermis (Fig. 1P). Severemuscle attachment defects occurred in 30.3% of elmo19F3/KO

mutant embryos (Fig. 1Q; supplementary material Table S1).

To confirm whether the muscle detachment phenotype was dueto a requirement for Elmo in the muscle or tendon cells, we

performed tissue-specific rescue experiments by expressing afull-length elmo cDNA (UAS-elmo-FL) with the appropriateGAL4 drivers. The extent of muscle detachment rescue for each

embryo was categorized according to the classes in Fig. 1M–P. Inthe zygotic elmo-null genetic background, sole expression ofElmo-FL in the tendon cells using sr-GAL4 did not improvemuscle attachment compared with elmo mutants alone. In

contrast, the muscle detachment phenotypes were efficientlyrescued by the muscle-specific expression of Elmo using eitherthe muscle-specific mef2-GAL4 or mesoderm-specific 24B-

GAL4 drivers. Although the elmo mutant embryos were notcompletely rescued to WT using either of the muscle GAL4drivers, no severe muscle defects were observed, accompanied by

the ,40% increase in embryos with WT muscle shape. Theseresults indicate that Elmo, like Dim7, functions within musclecells to mediate muscle attachment.

Elmo constructs that removed regions of the protein predicted

to be important for function were introduced into elmo mutants toreveal the relative contribution of each domain in fly viability.The ubiquitous expression of Elmo using the actin-GAL4 driver

fully rescued lethal elmo mutations to adult viability in twoheterozygous elmo mutant backgrounds, elmo9F4/19F3 or elmoPB/19F3

(Geisbrecht et al., 2008). The expression of deleted versions of

Elmo in elmo mutants (ElmoDN, ElmoDC, or ElmoDPH) failed torescue lethality, indicating indispensable roles of either the N- or C-terminal regions in Elmo for fly survival. In contrast, a small portion

(7–12%) of adult flies eclosed when the ElmoDPxxP truncation wasexpressed in an elmo mutant background (Fig. 1Q; supplementarymaterial Table S1).

We extended these structure/function studies into the

musculature with the expectation that we could uncover thedomains of Elmo essential in myogenesis. The functionalcontribution of the N- or C-terminal regions of Elmo was

determined by expressing either UAS-elmoDN or UAS-elmoDC

under control of mef2-GAL4 or 24B-GAL4 in elmo mutants(Fig. 1Q; supplementary material Table S1). Muscle-specificoverexpression of any of the indicated Elmo deletion constructs

did not reveal obvious embryonic muscle patterning defects,suggesting that Elmo did not induce dominant effects in themusculature (data not shown). Expression of the N-terminal

region of Elmo only (ElmoDC) had no effect on rescuing theattachment defects, whereas reintroduction of the Elmo C-terminal region (ElmoDN) provided partial rescue. Using the

same experimental assay to evaluate the role of Elmo in myoblastfusion, we observed that the ElmoDN-truncated version providedpartial rescue of elmo-triggered myoblast fusion defects, whereas

the ElmoDC did not (supplementary material Fig. S2; Table S2).Thus, it appears that the C-terminal portion of Elmo containsinformation to direct both early myoblast fusion events(Balagopalan et al., 2006), and subsequent muscle attachment

events in myogenesis.

Dim7 genetically and physically interacts with Elmo both invitro and in vivo

The identification of Dim7 as a possible Elmo-binding partner inour in vivo proteomics approach and the similarities in the msk andelmo embryonic phenotypes raise the possibility that a Dim7–

Elmo complex could function in the musculature. To confirm abiochemical interaction between Elmo and Dim7, we firsttransfected S2 cells with a FLAG-tagged version of Elmo-FL

and subjected the resulting lysates to a GST pulldown assay using abacterially expressed GST–Dim7 fusion protein. Elmo protein wasdetected in a complex with GST–Dim7, but not GST alone(Fig. 2A). Our rescue results in Fig. 1 suggest that the C-terminal

region of Elmo is essential to mediate muscle attachment.Consistent with this, S2-transfected ElmoDC–FLAG could notbe found in a complex with GST–Dim7 (Fig. 2B).

To confirm an in vivo physical interaction between Elmo andDim7, third instar larval lysates expressing an Elmo–YFP fusionprotein in the musculature were incubated with GST alone orGST–Dim7 beads. As shown in Fig. 2C, both endogenous Elmo

and the higher molecular mass Elmo–YFP protein were detectedin GST–Dim7 pulldown experiments. Using these same Elmo–YFP lysates, western blotting confirmed the presence of Dim7 in

anti-Elmo immunoisolated complexes (Fig. 2D). To verify thisDim7–Elmo interaction without overexpression of Elmo proteins,we performed immunoprecipitations (IPs) with endogenous Elmo

or Dim7 protein lysates from WT embryos or larvae. Dim7 wasdetected in anti-Elmo immunoisolated complexes from bothembryos and larval lysates. Elmo protein was also detected upon

GST pulldown of Dim7 (supplementary material Fig. S3).

In vitro studies with human ELMO1 reveal that mutations in theEAD domain (ELMO1L202E/I204D/L205E) unleash an autoinhibitoryinteraction in the native protein, similar to the conformational

changes observed in Elmo upon Dock180 binding (Patel et al.,2010). We used site-directed mutagenesis to recapitulate mutationsthat prevent the autoinhibitory interaction between the EID and

EAD (hereafter referred to as ElmoEDE) domains confirmed inELMO1 (supplementary material Fig. S1). After the creation oftransgenic flies expressing UAS–ElmoEDE–YFP, we tested

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whether this ‘activated’ version of Elmo binds Dim7. Larval

lysates expressing ElmoEDE in the musculature were subjected to

GST–Dim7 pulldown assays (Fig. 2C) or anti-Elmo IPs (Fig. 2D)

and in all experiments, a Dim7–ElmoEDE interaction was

detected. These data suggest that Dim7 can bind Elmo in either

the open or closed conformation.

We took advantage of genetic interaction analyses to

determine whether Dim7 functions with the Elmo–Mbc

complex for stable muscle attachment. Similar to the

quantification method used in the experiments in Fig. 1Q, each

genotype was categorized into three phenotypic classes on the

basis of the severity of the muscle detachment phenotype: WT

(Fig. 2F), minor attachment defects (Fig. 2G), or severe

attachment defects (Fig. 2H). In elmo19F3/KO mutant embryos,

only 21% of embryos had either minor or severe muscle

attachment defects (Fig. 2I; supplementary material Table S1).

However, the number of affected individuals increased to 54% in

elmo19F3/KO embryos that also lacked one copy of the msk allele.

In the converse experiment, removal of one copy of elmo in a

homozygous msk4/msk5 mutant background (74% detached

Fig. 2. Elmo and Dim7 biochemical and

genetic interactions. (A) GST pulldowns of

S2 cells transfected with ElmoFL–FLAG or

ElmoDC–FLAG constructs. Anti-FLAG

detects ElmoFL-FLAG, but not ElmoDC-

FLAG in a complex with GST–Dim7.

(B) Loading control for A. (C,D) Lysates of

larval expressing Elmo–YFP or ElmoEDE–

YFP were incubated with either GST alone

or GST–Dim7 beads (C) or anti-Elmo

antibodies for immunoisolation (D). Western

blotting using anti-Elmo (C) or anti-Dim7

(D) antibodies reveal that both endogenous

Elmo and YFP-tagged Elmo proteins are

associated with Dim7. (E) Loading controls

for C and D. (F–I) Classification of muscle

detachment in stage 16–17 embryos.

(F) Class I: WT and WT-like; (G) Class II:

minor attachment defects; (H) Class III:

severe attachment defects. (I) Quantification

of embryonic muscle attachment defects

shows that embryos heterozygous for msk

(msk/+) enhance the attachment defects in

homozygous elmo mutants, whereas loss of

one copy of elmo or mbc enhances muscle

detachment in msk/msk mutants.

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muscles) moderately enhanced the muscle detachment phenotype

compared to msk mutants alone (55% detached muscles). An

increase in the number of embryos with detached muscles was

also observed upon reduction of mbc in a msk mutant background

(73% compared with 55% with detached muscles). This data

strongly suggests that msk genetically interacts with the Elmo–

Mbc complex in the processes of muscle attachment.

MAS localization of Dim7, Elmo and Mbc during embryonicand larval myogenesis

We utilized previously generated anti-Elmo antibodies (Geisbrecht,

et al., 2008) to examine the subcellular distribution of Elmo protein

in embryogenesis. These immunostainings revealed a cytoplasmic

distribution and faint muscle attachment site accumulation of Elmo

in developing myofibers (supplementary material Fig. S4). In larval

muscles, this same Elmo staining pattern persisted and we

additionally detected Elmo protein in the nucleus. All anti-Elmo

staining in the musculature appeared specific as this signal was lost

in elmoKO mutants (supplementary material Fig. S4). Therefore, we

examined the full-length (Elmo–YFP) and activated YFP versions

of Elmo (ElmoEDE–YFP) to discern whether we could observe a

change in the spatiotemporal localization of Elmo in myogenesis.

The ElmoEDE–YFP fusion protein was mainly in the myoplasm

and weakly observed at the ends of a few myofibers in stage 14

embryos (Fig. 3A–A9). Activated Elmo began to accumulate at the

ends of the ventral muscle fibers in stage 15, when muscle

migration is complete and the establishment of adhesion junctions

between the muscle and tendon cells is initiated (Fig. 3B). After

stage 16, ElmoEDE–YFP overlapped with bPS integrin at the

MASs (Fig. 3C). Analysis of Elmo–YFP revealed a similar

distribution except that basal Elmo was not apparent until after

stage 15 (supplementary material Fig. S4). Since this pattern of

Elmo localization mirrors that of Dim7 localization in myogenesis

(Liu and Geisbrecht, 2011), we sought to extend our

immunolocalization analysis in the larval musculature to

determine whether Elmo and Dim7 still accumulate at the MASs

Fig. 3. In vivo detection of a Dim7–Elmo complex at MASs in developing and mature muscles. (A–C9) Muscle-specific expression of ElmoEDE–YFP in two

hemisegments of the embryonic ventral muscles. ElmoEDE protein, barely detectable in stage 14 embryos (A,A9), accumulates at the ends of myofibers in stage 15

(B,B9) or stage 16 (C,C9) embryos. (D–G9) Filleted L3 individuals showing Dim7 accumulation at MASs, revealed using an antibody against Dim7 (D,D9) or by

expressing a YFP–Dim7 fusion protein (E,E9). Elmo protein persists at MASs into late larval stages (F–G9). (H–Q0) Duolink in situ PLA in WT embryos or a third instar

larva reveal that Elmo and Dim7 proteins are found in close proximity at the sites of muscle attachment. Virtually no signal is observed in control samples (H,K,N,O).

(H–M9) Three segments of the embryonic ventral muscles are shown either in lateral (H,H9,I,I9,J,J9) or dorsal views (K,K9,L,L9,M,M9). Weak duolink signal

between Elmo and Dim7 is observed at stage 15 (I,I9,L,L9), and the signal intensifies from stage 16 onwards (J,J9, M,M9). (N–Q0) Phase-contrast and fluorescence images

are shown for L3 muscle fillets where intense Duolink signal is observed between Elmo and Dim7 at larval MASs (P–Q0). Arrowheads indicate MAS.

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in actively contracting muscles. Continual MAS localization of

Dim7 (Fig. 3D–E9) and Elmo (Fig. 3F–G9) persisted in all larval

stages. Examination of Dim7, Elmo or Mbc distribution within the

body wall muscles of larvae revealed they were all enriched at the

Z-disc also (supplementary material Fig. S5).

To confirm an in vivo association between Elmo and Dim7 within

muscle tissue, we took advantage of the in situ proximity ligation

assay (PLA) technology, or Duolink assay. The PLA recognizes the

potential interaction of endogenous proteins using antibodies to

detect proteins in close proximity to one another (,40 nm). As this

technique was new in our hands, a series of control experiments was

performed to ensure signal specificity. First, omission of either or

both primary antibodies and exposure to secondary antibodies

conjugated with complementary PLA probes and amplification

reagents resulted in non-specific signal in embryos

(Fig. 3H,H9,K,K9) or larval tissue (Fig. 3N–O0; supplementary

material Fig. S6). Incubation of an anti-Elmo with an antibody

generated against the transcription factor Mef2 resulted in faint

signal in a few nuclei (supplementary material Fig. S6). In contrast,

the in vivo Dim7–Elmo Duolink signal was weakly detected at the

embryonic MASs at stage 15 (Fig. 3I,I9,L,L9) and remained strong

after the completion of muscle attachment (Fig. 3J,J9,M,M9). In

actively contracting larval muscles, Elmo–Dim7 staining was

apparent at the MTJs (Fig. 3P–Q0; supplementary material Fig.

S6). These data are consistent with a Dim7–Elmo complex that

functions at the MASs to maintain strong and cohesive MTJ.

Dim7 functions upstream of the Elmo–Mbc complex formuscle attachment

To determine where Dim7 resides within the Elmo–MbcRRac

pathway in the attachment of embryonic muscles, we took

advantage of a previous strategy for analyzing this tripartite

complex in the Drosophila eye (Geisbrecht et al., 2008). A rough

eye phenotype induced by Elmo–Mbc overexpression is

suppressed upon removal of rac1J11 and rac2D, resulting in a

WT eye morphology, and is consistent with Rac functioning

downstream of Elmo–Mbc. To confirm this signaling paradigm

in the somatic musculature and extend this strategy to determine

where Dim7 functions, we overexpressed both Elmo and Mbc

together in muscle and simultaneously reduced one gene copy of

endogenous rac1, rac2 or msk. Overexpression of either Elmo

alone or Mbc alone resulted in a WT-like muscle pattern

(Geisbrecht et al., 2008), similar to mef2-GAL4/+ (Fig. 4A).

Both myoblast fusion defects and muscle detachment phenotypes

were prevalent upon muscle-specific overexpression of the

Elmo–Mbc complex (Fig. 4B). Muscle detachment was largely

rescued to WT in a Drac1, rac2/+ genetic background (Fig. 4C).

In contrast, there was no suppression of muscle attachment

defects as a result of expression of this RacGEF complex upon

removal of one copy of the msk4 allele (Fig. 4D,K). These data

suggest that Dim7 functions either upstream or parallel to the

Elmo–Mbc complex.

We next tested whether the distribution of pMAPK

(Fig. 4L,L9) or pFAK protein (Fig. 4P,P9) was altered upon

activated-Rac-induced muscle detachment. In embryos with an

excess of both Elmo and Mbc in the musculature, the number of

pMAPK-positive tendon cells was reduced (Fig. 4M,M9), while

pFAK was still properly localized (Fig. 4Q,Q9). Embryos of the

genotype mef2:UAS-elmo,UAS-mbc; rac1J11, rac22/+ exhibited

WT pMAPK and pFAK expression patterns with six or seven

aligned pMAPK(+) tendon cell nuclei in each hemisegment

border (Fig. 4N,N9), and larger, extended pFAK-enriched MASs

Fig. 4. The muscle-specific Elmo–MbcRRac signaling module non-autonomously signals to maintain tendon cell identity through pMAPK.

(A–D) Representative embryos detailing the lateral musculature (TM; red) and tendon cell nuclei (pMAPK; green) in stage 16–17 embryos. Muscle-specific

coexpression of Mbc and Elmo result in severe muscle attachment defects with smaller MASs (B; arrows) than mef2-GAL4/+ control embryos (A). Removal of

one copy of endogenous rac1J1rac2D(C), but not msk4 (D), restores normal muscle attachment. (E–K) For quantification (K), the muscles in hemisegments A1–4

in each embryo (n5200) were scored as WT (E,H); minor (F,I); or severe muscle detachment (G,J). (L–S9) High magnification of two hemisegments of the

embryonic ventral musculature (TM; red) co-stained with the muscle or tendon cell markers, pFAK and pMAPK, respectively (green). In mef2-GAL4 controls,

pFAK properly localizes to MASs (P,P9) with 6–7 pMAPK(+) tendon cells at the segmental border (L,L9). An excess of Elmo and Mbc in muscles results in

smaller MASs, indicated by pFAK staining (Q,Q9) and a decrease in the number of pMAPK(+) cells (M,M9). The pFAK and pMAPK expression patterns are

rescued to WT with the decrease of endogenous rac1 and rac2 (N,N9, R,R9), whereas they remain unaltered upon loss of one copy of msk (O,O9, S,S9).

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(Fig. 4R,R9), demonstrating that rescue of the muscle attachment

defects also rescued tendon cell identity. However, a reduction

in Dim7 levels (mef2:UAS-elmo,UAS-mbc; msk4/+) was

not capable of suppressing aberrant pMAPK accumulation

(Fig. 4O,O9) or pFAK localization (Fig. 4S,S9). Activation

of Rac through the Elmo–Mbc complex phenocopies the loss

of pMAPK in the tendon cells observed upon overexpression of

Dim7 in the musculature (Liu and Geisbrecht, 2011), suggesting

that the Elmo–Mbc complex may function with Dim7 to mediate

tendon cell identity.

The above genetic experiments suggested that Dim7 is not

downstream of Elmo, so we tested whether Dim7 functions

upstream of the Elmo–MbcRRac signaling pathway by

attempting to answer two independent questions: (1) is the

muscle detachment phenotype associated with the muscle-

specific overexpression of Dim7 suppressed by reducing

endogenous levels of Mbc?; (2) can the msk muscle

detachment phenotype be rescued by overexpression of putative

downstream components? Consistent with our previously

published results (Liu and Geisbrecht, 2011), excess Dim7 in

the musculature, from the early mesoderm driver twist-GAL4,

perturbs the ability of the muscles to firmly adhere to the tendon

cells (Fig. 5A). However, further removing one copy of mbc

largely suppressed the muscle attachment defects, revealed by a

decrease in embryos with severe muscle attachment defects from

,30% to 3% (Fig. 5B,C). These data suggest that Dim7 may act

upstream of the Elmo–MbcRRac signaling pathway. Further

support for this linear model was revealed by rescue experiments

in which we expressed putative downstream proteins in an

attempt to ameliorate muscle detachment in msk4/4 mutants

(Fig. 5D). The introduction of exogenous Elmo (Fig. 5E) or the

Elmo–Mbc complex (Fig. 5F) partially rescued the muscle

detachment phenotype associated with mutations in msk,

although more complete rescue was observed with the latter

GEF complex (Fig. 5H; supplementary material Fig. S7). If the

Elmo–Mbc complex upregulates Rac activity within the

Fig. 5. Genetic manipulation places Dim7 upstream of the Elmo–Mbc complex. (A,B,D–G) MHC staining of stage 16–17 embryos. (A–C) Excess Dim7

expression in myofibers results in the separation of muscles from smaller MASs (A). This muscle detachment phenotype is suppressed by removal of one copy of

mbcD11.2 (B,C). (D–H) Expression of Elmo–MbcRRac signaling components rescues the muscle defects in msk mutants. The severe attachment defects observed

in msk4/msk4 embryos (D) are partially restored upon muscle-specific expression of Elmo (E). Improved restoration of muscle attachment is observed by

introduction of the Elmo–Mbc complex (F) or RacV12 (G). (H) Bar graph quantifying the rescue of muscle attachment defects in msk4/4 mutant embryos upon

muscle-specific expression of Elmo, the Elmo–Mbc complex, the ElmoEDE variant or RacV12.

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musculature, expression of activated Rac would be expected to

also rescue msk mutants. To circumvent the complete block inmyoblast fusion upon excess Rac activation using mef2-GAL4,we reduced the levels of activated Rac being expressed in early

myogenesis using the weaker sticks and stones (sns)-GAL4driver that is expressed in a subset of myoblasts (Kocherlakotaet al., 2008). Although occasional missing muscle fibers werepresent in embryos of the genotype msk4/4, sns-GAL4::UAS-

RacV12, presumably due to incomplete fusion events, the muscleattachment defects were largely rescued (Fig. 5G,H). We did notobserve complete rescue of the muscle detachment phenotype in

msk4/4 mutants using this assay. Excess levels of Elmo–Mbc,RacV12 or Dim7 alone resulted in detached muscles, suggestingthat protein levels or regulation of protein activity is precisely

controlled in establishment of the muscle–tendon junction. Thesedata, taken together, indicate a linear pathway in which Dim7acts upstream of the Elmo–MbcRRac signaling to mediate

muscle attachment.

The recruitment of activated Elmo to the MASs requiresDim7

The open, activated version of human Elmo

(ELMO1L202E/I204D/L205E, or ElmoEDE) promotes cell elongationin cultured cells (Patel et al., 2010). To extend these studies in vivo

and reveal how uninhibited Elmo may influence muscle

attachment, we expressed this activated version of Drosophila

Elmo using mef2-GAL4. Strikingly, muscle-specific expression ofElmoEDE–YFP showed high levels of accumulation at larval

MASs (Fig. 6B–B0), whereas native ElmoFL–YFP retained abroader cytoplasmic distribution within the myofiber (Fig. 6A–A0). Our hypothesis that ElmoEDE functions as an activated Elmo

variant is supported by two lines of experimentation. First, 70% ofL3 larvae with excess ElmoEDE had at least a few detachedmuscle fibers. Furthermore, ElmoEDE mimicked rescue of themuscle detachment phenotype in msk mutants to the same extent as

the Elmo–Mbc complex and better than ElmoFL alone (Fig. 5H).This is the first in vivo evidence in any tissue that Elmolocalization and/or activity could be regulated as a result of an

autoinhibitory switch.

We next sought to determine whether the upstream Dim7 isresponsible for the enrichment of activated Elmo to larval MASs.Upon loss of Dim7 by RNAi, the amount of ElmoEDE–YFP was

decreased at the ends of muscle fibers, independent of theirattachment state (Fig. 6C–C0). The reduction in activated Elmoappeared to be specific to a decrease in Dim7 as depletion of the

MAS protein Zasp52 had no effect on ElmoEDE–YFPlocalization at the ends of muscles (Fig. 6D–D0). To control fordifferences in data acquisition, we took two approaches. First,

confocal micrographs of control and msk RNAi knockdownlarvae were acquired simultaneously using the same microscopesettings for direct comparison (supplementary material Fig. S8).

Subsequent quantification of muscles in individual larvaerevealed a 40% decrease in the amount of ElmoEDE at theends of the muscles (Fig. 6E).

We examined global ElmoEDE protein levels by western blot

analysis using anti-YFP or anti-Elmo antibodies after boilingwhole larvae directly in SDS-loading buffer to eliminate thepotential loss of ElmoEDE localization with membrane-anchored

proteins. The mef2-GAL4/+ control and Zasp knockdownanimals exhibited similar levels of ElmoEDE (Fig. 6F,G),whereas overall ElmoEDE levels were largely reduced in

larvae with decreased Dim7 protein levels. These results

suggest that Dim7 is essential for the localization of activated

Elmo and/or ElmoEDE protein that is not anchored to the MASs,

which may get targeted for degradation in the myoplasm.

Integrins function upstream of Dim7–Elmo

Three pieces of evidence triggered us to investigate the

possibility that the Dim7–Elmo complex could be downstream

of integrins in MAS maintenance. First, msk was uncovered in a

genetic screen as a suppressor of a gain-of-function wing

blistering phenotype caused by mutations in inflated (if), which

encodes the aPS2 integrin subunit (Baker et al., 2002). Second,

integrins are well characterized as the major receptor complex

essential for both adhesion and signaling in muscle attachment

(Carmignac and Durbeej, 2012; Bozyczko et al., 1989). Finally,

both Dim7 and ElmoEDE become enriched at the sites of integrin

localization in the MASs and this localization persists throughout

active larval muscle contraction (Fig. 3) (Liu and Geisbrecht,

2011).

We first tested whether the localization of Dim7 protein was

altered upon disruption of the integrin adhesome, using a muscle-

specific RNAi knockdown strategy. Loss of aPS2, bPS, or ILK all

resulted in embryonic or first instar larval lethality using the mef2

or 24B GAL4 drivers. To circumvent this early lethality, we

recombined mef2-GAL4 with the temperature-sensitive GAL80ts

repressor (Lee and Luo, 1999) and mated these flies with the

appropriate UAS-RNAi line. The resulting progeny were raised at

the non-permissive temperature (18 C) throughout embryogenesis

to repress leaky GAL4 expression and then shifted to the

permissive temperature (29 C) to initiate RNAi induction at the

end of the first instar larval stage. As expected (Liu and Geisbrecht,

2011), filleted L3 larvae with decreased levels of if, mys or ILK

RNAi, all exhibited muscle attachment defects (Fig. 7B–D).

However, the localization of Dim7 was differentially affected in

these different genotypes. In control animals (mef2-GAL4,

GAL80ts) Dim7 accumulated at the ends of muscles (Fig. 7A9–

A0). The subcellular localization of Dim7 was dramatically altered

upon loss of aPS2 (Fig. 7B–B0) or bPS (Fig. 7C9–C0) where it was

found at high levels in the nuclei rather than being tethered to the

MASs (Fig. 7C). Knockdown of ILK had no effect on the

maintenance of Dim7 at the cell periphery in both attached and

detached muscles in these larvae (Fig. 7D9,D0) or in ilk mutant

embryos with detached muscles (supplementary material Fig. S8).

These results suggest that integrins or integrin-associated proteins

(exclusive of ILK) are responsible for tethering Dim7 to the ends

of the muscles.

The ability of Dim7 to control ElmoEDE accumulation

(Fig. 6) or integrins to mediate Dim7 localization (Fig. 7) at

the MASs obviated the need to examine whether integrins

modulate ElmoEDE localization. Inclusion of the Gal80ts

element had no effect on the accumulation of ElmoEDE to the

ends of myofibers (Fig. 8A,A9). The MAS accumulation of

ElmoEDE was attenuated in larvae with low levels of either the

aPS2 (Fig. 8B,B9) or bPS (Fig. 8C,C9). Consistent with the

results obtained for Dim7 localization, loss of ILK did not alter

ElmoEDE protein accumulation at the ends of either attached or

detached muscles (Fig. 8D,D9). Loss of Dystrophin (Dys) or

FAK had no effect on ElmoEDE MAS accumulation, suggesting

that ElmoEDE localization is integrin dependent (supplementary

material Fig. S8).

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DiscussionWe previously showed that Dim7 localizes to developing

muscle–tendon insertion sites and removal of Dim7 has severe

consequences in muscle attachment maintenance (Liu and

Geisbrecht, 2011). Studies herein extend these observations to

elucidate the functional contribution of Dim7–Elmo in regulating

Drosophila muscle attachment. Our results show that Dim7 is an

upstream adaptor protein that recruits Elmo in response to

integrin adhesion and/or signaling. Thus, we propose that the

spatial and temporal regulation of Elmo-Mbc activity results in

regulation of the Rac-mediated actin cytoskeleton changes at the

MASs.

The Dim7–Elmo complex functions in late embryonicmyogenesis for stable establishment of MASs

The ‘myospheroid’ phenotype in elmo or msk mutants resembles

attachment defects first characterized in mutated genes that

encode integrins, ILK and Talin, and is not due to earlier

developmental defects in myogenesis. A similar number of cells

expressing the muscle differentiation factor DMef2 was present

in elmo or msk mutants, indicating that muscle specification was

not affected (Geisbrecht et al., 2008; Liu and Geisbrecht, 2011).

Mutations in genes essential for muscle migration and targeting

also lead to detached muscles. For example, in kon/perd or grip

mutants, the early arrest of migrating myotubes resulting from

defective migration eventually leads to a linkage failure between

the muscle and tendon cells (Estrada et al., 2007; Schnorrer et al.,

2007; Swan et al., 2006; Swan et al., 2004). In mutant embryos

with reduced levels of Elmo or Dim7, the muscle detachment

phenotype did not appear to result from muscle migration defects.

First, the spatiotemporal accumulation of Elmo and Dim7 is

developmentally regulated. Both proteins are not detected at the

leading edges of migrating muscles, but begin to accumulate at

MASs after stage 15. Second, we did not observe a failure of

Fig. 6. MAS localization of ElmoEDE is Dim7 dependent in the larval musculature. (A–D-) F-actin (phalloidin; red) labels muscles present in one-half of a larval

fillet, while the localization of Elmo protein is detected by YFP fusion proteins driven by mef2-GAL4. Expression of C-terminally fused ElmoFL–YFP is broadly

distributed in the cytoplasm of muscle fibers (A–A-), whereas an ElmoEDE–YFP fusion protein is largely enriched at the MASs (B–B-). Accumulation of ElmoEDE at the

MASs is reduced in mef2-GAL4::UAS-elmoEDE–YFP, UAS-mskRNAi animals (C–C-), but unaffected in mef2-GAL4::UAS-elmoEDE–YFP, UAS-zaspRNAi (D–D-).

(E) Quantification of ElmoEDE–YFP accumulation at the ends of attached muscles in controls (mef2-GAL4::UAS-elmoEDE–YFP) and Dim7 or Zasp RNAi knockdown

larvae. The localization of ElmoEDE was determined at the ends of the same ten muscles (see Materials and Methods) in every hemisegment of dissected and

photographed fillets and displayed as the percentage of overall ElmoEDE accumulation. (F,G) Western blots and quantification of representative blots of total Elmo protein

from control and knockdown third instar larvae. Detected by anti-YFP (F) or anti-Elmo antibody (G), ElmoEDE levels in Dim7 knockdown larvae is significantly

reduced compared with control or Zasp52 knockdown larvae. Anti-a-tubulin staining was used as protein loading control.

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Fig. 7. Loss of integrins redistributes Dim7 from the ends of muscles to myofiber nuclei. (A–D-) Confocal micrographs of the L3 musculature to

show the repeated sarcomeric structure of actin filaments (phalloidin; red) and the subcellular localization of Dim7 (green). Severe muscle detachment phenotypes

are observed upon knockdown of PSa2 integrin, PSb integrin or ILK levels (B–D-). In control mef2-GAL4, GAL80ts larvae, Dim7 is enriched at the MASs

(A–A-). Upon loss of either integrin heterodimer subunit, Dim7 disappears from the muscle ends and is present at high levels in the myofiber nuclei

(B–C-). However, in muscles with decreased ILK, Dim7 is still tethered to the cell periphery, even in detached muscles (D–D-).

Fig. 8. Integrins are required for the localization of activated Elmo. (A–D9) Muscles in third instar larval were labeled with phalloidin to visualize

F-actin (red) along with the activated Elmo fusion protein (green). Compared with localization of ElmoEDE at the MASs (A,A9), this accumulation at the muscles

ends is reduced in PSa2 integrin (B,B9) or PSb integrin (C,C9) knockdown myofibers. Knockdown of ILK levels have no effect on ElmoEDE levels (D,D9).

(E) The pixel intensity of ElmoEDE in the indicated genetic backgrounds are measured across muscle 7. Note the levels of ElmoEDE accumulation at the

attachment sites (arrowheads in A–D9) upon knockdown of integrins are approximately half of the levels observed in controls.

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muscle ends contacting their corresponding attachment sites inelmo or msk mutants at late stage 15, when muscle migration wasalmost complete (Liu and Geisbrecht, 2011).

The recruitment of active Elmo protein to the MAS by Dim7

Both membrane localization and Rac-dependent cell spreading ofthe uninhibited, active version of Elmo is enhanced comparedwith native WT Elmo in cultured mammalian cells (Patel, et al.,

2010). These in vitro results are in agreement with our in vivo

analysis, which found ElmoEDE is enriched at larval muscle endscompared with the poor accumulation of full-length Elmo–YFP.This may reflect a potential regulatory mechanism controling the

subcellular localization of Elmo from the cytoplasm to themuscle ends upon the release of Elmo autoinhibition. Withindifferent cells or tissues, various proteins may regulate Elmo

localization to the cell periphery, or other sites where active Elmois needed. In cultured mammalian epithelial cells, membranerecruitment of the Elmo–Dock180 complex is dependent on

active RhoG for cell spreading (Katoh and Negishi, 2003).Consistent with a functional role for membrane-targeted Elmo,active Elmo promotes cell elongation in HeLa cells when co-

expressed with RhoG (Patel et al., 2010).

Our data argues that adaptor proteins may be required in musclecells for activated Elmo membrane recruitment. Decreased levelsof ElmoEDE are observed at the polarized ends of muscle insertionsites when Dim7 levels are decreased. It is still not clear if Dim7

binding is required for the conformational change that results inElmo activation or if an activated Dim7–Elmo complex alreadyexists within the cell and is recruited as a complex upon integrin

activation. Furthermore, we do not observe a complete loss ofElmoEDE protein levels, suggesting that either Dim7 proteinlevels are not depleted enough or other proteins in addition to

Dim7 play a role in Elmo membrane recruitment. Alternatively,post-translational modification(s), such as phosphorylation, couldbe an additional mechanism for the relief of Elmo autoinhibition.Thus, we conclude that in muscle, Dim7 is an essential adaptor

protein for the polarized membrane localization of active Elmo orthe active Elmo–Mbc complex downstream of integrin signalingpathway.

Integrins function upstream of the Dim7–Elmo complex

What is the relationship between the integrin adhesome and theDim7–Elmo complex? We propose two possibilities, which are notmutually exclusive. One is that the Dim7–Elmo–Mbc complex

assembles at MASs through integrin-mediated ‘outside-in’signaling. Upon ligand binding to ECM molecules, integrinactivation results in Dim7–Elmo–Mbc complex localization forthe spatiotemporal regulation of Rac activity to maintain dynamic

actin filament adhesion at the MASs. We predict that localizationof activated Elmo to the MASs is a prerequisite regulatorymechanism for actin cytoskeleton remodeling through Rac to

maintain stable attachments. This hypothesis is supported by threelines of evidence: (1) muscle attachment defects upon loss of Dim7or Elmo are only observed after the establishment of the integrin

adhesion complex and onset of muscle contraction; (2) muscledetachment in msk mutants can be rescued by expressing lowlevels of activated Rac; and (3) the enrichment of Dim7 and

ElmoEDE proteins at the ends of muscle fibers is greatly reducedin integrin-deficient larvae.

The other possibility is that accumulation of the Dim7–Elmocomplex to the ends of muscles regulates ‘inside-out’ signaling to

dynamically regulate integrin affinity for strong ligand binding and

stable muscle attachments. Previously, we reported that Dim7 acts

upstream of the Vein–Egfr signaling pathway in muscle to tendon

cell signaling (Liu and Geisbrecht, 2011). Combined with previous

reports that muscle-specific Vein secretion is dependent on the

adhesive role of bPS integrin (Martin-Bermudo, 2000; Yarnitzky

et al., 1997), the Dim7–Elmo complex may be internally required

for integrins to regulate Vein secretion. A decrease in Vein–Egfr

signaling and loss of tendon cell terminal fate results in a reduction

in ECM secretion and weakened integrin attachment to the ECM.

This is consistent with the observation that msk or elmo mutants

phenocopy embryos with reduced or excessive amounts of the

aPSbPS integrin complex, where pointed muscle ends result in

smaller muscle attachments. Future studies analyzing Dim7–

Elmo–Mbc complex localization and function in the background

of integrin deletion constructs which separate the ‘inside-out’ and

‘outside-in’ signaling pathways will be essential to uncover more

detailed molecular mechanisms.

What is the relationship between the Dim7–Elmo–MbcRRac

signaling pathway and the integrin-mediated adhesome complex

assembly (including Talin and the IPP complex)? We propose

that actin filaments within the muscle cell are anchored to the

muscle cell membrane through the IPP complex, whereas

regulation of MAS–actin remodeling is controlled by the

Dim7–Elmo–MbcRRac pathway. Our data suggests that these

two complexes assemble independently at the muscle ends. In

msk mutant embryos, both ILK and Talin properly accumulate at

the MASs, suggesting that Dim7 is not responsible for their

localization (Liu and Geisbrecht, 2011). Similarly, we can still

detect both MAS-enriched Dim7 and active Elmo at two ends of

the muscles in ILK-deficient larvae, even in fully detached

muscles. In a vertebrate cell culture model, Elmo2 was found to

physically interact with ILK for the establishment for cell

polarity (Ho and Dagnino, 2012; Ho et al., 2009). Thus, it is

possible that our approaches have not fully knocked down Ilk

levels or that the Dim7–Elmo recruitment by Ilk is redundant

with another attachment site protein. Alternatively, an upstream

scaffold protein may function to recruit both the IPP and Dim7–

Elmo complexes to the MASs. It is probable that these two

complexes are temporally regulated in embryogenesis, where the

actin remodeling complex is not needed until initial muscle–

tendon initiation has been established.

Materials and MethodsFly genetics

Fly stocks were raised on standard cornmeal medium at 25 C unless indicatedotherwise. The y1,w1118 strain was used as the wild-type control. The following flystocks and/or alleles were used in this study: msk4/TM3-lacZ [a gift from LizabethPerkins (Baker et al., 2002)]; mef2-GAL4 (Liu and Geisbrecht, 2011); sns-GAL4(Kocherlakota et al., 2008); mef2-GAL4, Gal80ts (Liu and Geisbrecht, 2012);UAS-elmo (Geisbrecht et al., 2008); UAS-mbc (Balagopalan et al., 2006); mef2-GAL4, rac1J11, rac22 (Geisbrecht et al., 2008); mbcD11.2/TM3-lacZ (Ericksonet al., 1997); mbcD11.2, twist-GAL4/TM3-lacZ (Biersmith et al., 2011); mef2-GAL4,msk4 (Liu and Geisbrecht, 2011). The following stocks were obtainedfrom the Bloomington Stock Center: y1w; msk5P{neoFRT}80B/TM3, P{ftz-

lacZ.ry+}TM3, Sb1 (BL-23879); sr-GAL4/TM6 (BL-26663); UAS-msk (BL-23944);UAS-mskRNAi (BL-27572); UAS-Zasp RNAi (BL-31561); UAS-mys RNA (BL-33642); UAS-if RNAi (BL-27544); UAS-ILK RNAi (BL-35374).

Immunostaining and microscopy

Embryos were collected on agar-apple juice plates and aged at 25 C. Rescueexperiments were conducted at 29 C unless otherwise specified. Forimmunostaining, embryos or dissected third instar larva were fixed with 4%formaldehyde and stained as described previously (Geisbrecht et al., 2008; LaBeau-DiMenna et al., 2012). Primary antibodies used were: anti-MHC (mouse 1:500;

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Susan Abmayr, The Stowers Institute for Medical Research, Kansas City, MO); anti-Elmo (1:3000 for western and 1:400 for immunochemistry) (Geisbrecht et al., 2008);

anti-Msk (rabbit 1:3000 for western and 1:400 for immunochemistry) (Lorenzenet al., 2001); anti-TM (rat 1:50; Babraham Institute, Cambridge, UK); bPS-integrin[mouse 1:50; Developmental Studies Hybridoma Bank (DSHB)]; anti-Tig (mouse1:1000) (Fogerty et al., 1994); anti-FAK[pY397] (rabbit 1:1000; Invitrogen); anti-

dpERK/MAPK (rabbit 1:100; Cell Signaling).

Secondary antibody used for tyramide enhancement was goat anti-rabbit-HRP(1:200, Jackson Laboratories). Secondary antibodies for fluorescent immunostaining

were Alexa Fluor 488 or Alexa Fluor 546 (1:400; Molecular Probes). Phalloidin 594(or 647) was used for F-actin labeling (Molecular Probes). Tyramide enhancementwas used to improve signal intenisty for anti-Msk and anti-dp-ERK-MAPK (Cell

Signaling). Fluorescence images were collected on an Olympus Fluoview300 andprocessed using Adobe Photoshop Elements 2.0.

In situ proximity ligation assay

DuolinkTM in situ proximity ligation assays (PLA) were performed to detect in

vivo interactions between Elmo and Dim7 in embryonic myogenesis until larvalstages. PLA provides a fluorescent signal when the target proteins are localized

within 40 nm. The freshly collected WT embryos and dissected third instar larvaare fixed, permeabilized and incubated with anti-Elmo and anti-Dim7 primaryantibodies followed by secondary antibodies conjugated to complementary PLA

probes. Duolink hybridization, ligation, amplification and detection media wereadministered according to the manufacturer’s instructions (Olink Biosciences).Fluorescence duo-link signals were collected on the Olympus Fluoview300 andprocessed using Adobe Photoshop Elements 2.0. For control samples, no primary

antibodies, but only secondary antibodies were added, followed by standardamplification, and detection methods.

Molecular biology and site-directed mutagenesis

All Elmo deletion constructs were generated using standard PCR-basedtechniques. For all FLAG-tagged constructs, sequences encoding the FLAG

epitope were added to the 39 end of the appropriate Elmo sequences, with a stopcodon engineered in the oligonucleotide after the tag. The ORF of the full lengthelmo cDNA (Geisbrecht et al., 2008) was cloned in the proper reading frame into

the Gateway entry vector (Drosophila GATEWAYTM cloning system, Invitrogen).The amino acid changes in the EID region of FL Elmo (L194E/I196D/L197E)were introduced via standard site-directed mutagenesis methods (QuikChange IISite-Directed Mutagenesis Kit, Agilent Technologies). ElmoFL-YFP and Elmo

EDE-YFP fusions were generated using the pTWV gateway destination vectors(pUAST-based vector, EYFP fusions at the C-terminal end) with the Drosophila

Gateway Cloning System according to the instruction manual.

Immunoblotting

For western blots with whole larva, five third instar larvae from each genotype

were directly boiled in 300 ml 26SDS running buffer [100 mM Tris-HCl, pH 6.8,200 mM dithiothreitol, 4% SDS (electrophoresis grade), 0.2% Bromophenol Blue,20% glycerol] for 10 minutes. 10 ml from each genotype was subjected to western

blotting and probed with anti-YFP (MBL International) or anti-Elmo antibody.Anti-a tubulin was used as the protein loading control.

AcknowledgementsWe thank our colleagues for the kind gifts of antibodies and fly stocksas indicated in Materials and Methods. We also thank the BloomingtonStock Center at Indiana University and the Vienna Drosophila RNAiCenter (VDRC) for fly stocks and the Iowa Developmental StudiesHybridoma Bank and Babraham Institute for antibodies. We aregrateful to Zongheng Wang for critical reading of the manuscript.

Author contributionsN.O. contributed to the execution of experimental data. Z.L. andE.R.G. contributed to the conception, design, execution andinterpretation of the data being published, and preparing the article.

FundingThis work was supported by the National Institutes of Health [grantnumber R01 AR060788 to E.R.G.]; and the University of Missouri,Kansas City Graduate Women’s Fellowship (GAF) to Z.L. Depositedin PMC for release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.132241/-/DC1

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